1. Field of the Invention
The present invention relates to a biomolecule analyzer which obtains statistical properties of a plurality of specific portions of a sample and an interaction between the portions.
2. Description of the Related Art
Concerning a confocal scanning type optical microscope, for example, there is an explanation in a document “Confocal Microscopy” T. Wilson (ed.) Academic press (London). As an explanation which mainly targets a biological sample, there is a document “Handbook of Biological confocal Microscopy” J. B. Pawley (ed.) Plenum Press (New York) or the like. Concerning fluorescent correlation spectroscopy, for example, there is an explanation in a document “Fluorescence correlation spectroscopy” R. Rigler, E. S. Elson (eds.) Springer (Berlin) or the like.
In the 1990s, there has been a rapid increase in studies on single molecule detection/imaging which uses fluorescence. For example, as single molecule detection methods, a document P. M. Goodwin etc. ACC. Chem. Res. (1996), Vol. 29, p 607 to 613, fluorescent correlation spectroscopy (FCS), and the like can be cited. According to the fluorescent correlation spectroscopy, fluorescence-labeled proteins or carrier particles are suspended in a solution within a visual field of a confocal laser microscope, a fluctuation in fluorescence intensity based on Brownian motion of such fine particles is analyzed to obtain an auto-correlation function, and the number, sizes or the like of target fine particles is estimated. For example, this technology is discussed in PCT National Publication No. 11-502608 or “Protein nucleic acid enzyme” by Masataka Kinjo, Vol. 44, No. 9, p 1431 to 1437 (1999).
Meanwhile, concerning the confocal scanning type microscope and the fluorescent correlation spectroscopy, some patent applications have been filed. For example, according to Jpn. Pat. Appln. KOKAI Publication No. 2001-194303, a laser beam is guided to a cylindrical lens, and an exciting light beam in a line shape which is not a spot light is accordingly generated to illuminate a sample. This line-shaped light beam is subjected to light scanning by a galvanometer mirror to move in a vertical direction of the cylindrical lens, thereby exciting the entire portion in an imaging area of the sample two-dimensionally. A fluorescent signal from the sample is converted into an image by a two-dimensional photodetector such as a CCD. By this method, molecules only of a translational diffusion speed lower than a scanning speed of the cylindrical lens are excited to display fluorescence on the image.
Additionally, according to Jpn. Pat. Appln. KOKAI Publication No. 08-43739 and Jpn. Pat. Appln. KOKAI Publication No. 2000-98245, at a scanning type optical microscope, fluorescence from a multiple-dyed sample is subjected to spectroscopy through a grating or a prism, and detected for each component wavelength by a plurality of photodetectors.
According to Jpn. Pat. Appln. KOKAI Publication No. 08-068694, fluorescent correlation analysis is carried out from a spatially separated portion by entering an optical fiber into a reaction container which receives a liquid sample. It is disclosed that one light beam is separated into a plurality by multiplexing, thereby detecting different fluorescence intensities from a plurality of reaction containers.
According to Jpn. Pat. Appln. KOKAI Publication No. 09-113448, a plurality of reaction containers are arrayed in a line shape, one laser beam is applied to pass through all the reaction containers, a condenser lens is arranged in a front part in each reaction container to generate one focus in each of all the containers, and fluorescent correlation analysis is simultaneously carried out for samples in the reaction containers.
According to Jpn. Pat. KOKAI Publication No. 10-206742, at a laser scanning type confocal optical microscope, observation images of different times are obtained by two independent scanning optical systems, and dynamic characteristics of a sample are obtained based on a position movement when the images are superimposed together.
According to conventionally executed fluorescent correlation spectroscopy, a fluctuation of fluorescence intensity is observed from fluorescent molecules present in a visual field, and a time-sequential signal is accordingly obtained to calculate an auto-correlation function. In this case, if the fluorescent molecules present in the visual field are only one type, by directly analyzing the obtained fluctuation in fluorescence intensity, it is possible to obtain information regarding a translational diffusion speed or the like of the fluorescent molecules. Moreover, even if the fluorescent molecules move or change a motion speed, it is possible to statistically obtain such changes. If there are two or more types of fluorescent molecules different from each other in emission wavelength in a sample, by executing wavelength separation, it is possible to obtain an auto-correlation or a cross-correlation of the fluorescent molecules. However, such is limited in the same visual field.
In the case of observing an actual biological cell, real-time observation of behaviors of desired molecules inside/outside the cell or inside/outside a cell nucleus, and obtaining of changes with time or local information regarding phenomena such as signal transmission in the cell, material transportation and cell division are necessary. According to the conventional fluorescent correlation spectroscopy, a state change or behaviors of a group of molecules can be understood. However, it is impossible to dynamically measure a state change of a desired portion inside/outside the cell.
It is an object of the present invention to provide a biomolecule analyzer which can obtain various movements and changes of a target sample.